FIG. 3.

Regulation of GSH metabolism in mitochondrial redox mutants. (A) The wild-type glr1, trr2, trx3, glr1 trr2, and glr1 trr2 trx3 mutant strains were grown to an exponential phase in respiratory media (SGE) and the levels of total GSH and oxidized GSH (GSSG) were determined in cytosolic and mitochondrial fractions. Values shown are the means of three independent determinations and are expressed as an nmol/mg protein. There is no significant difference between the glr1 trr2 and glr1 trr2 trx3 mutants. (B) Redox Western blot analysis. The wild-type, glr1 trr2 and glr1 trr2 trx3 mutant strains expressing cytosol-, IMS- or matrix rxYFP were grown to an exponential phase in respiratory media (SGE). Cell extracts were separated on nonreducing SDS-PAGE gels and Westen blots probed with anti-GFP antibodies. Cells were treated with DTT to fully reduce the redox probes as indicated. Oxidized and reduced proteins are indicated. GSSG, oxidized glutathione; GSH, glutathione; IMS, mitochondrial intermembrane space; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; SGE, synthetic glycerol ethanol.